Microfossil extraction and preparation

[LabRatKing]

Hoping some of the pros here are willing to share microfossil prep techniques or suggestions to journal articles on the subject. I have been hunting conodonts and the like for quite some time, but the glacial acetic acid digestion and pan and scan techniques have failed me.

I experimented with HCl and H2SO4 in various concentrations, and even tried some ion exchange extractions ( which work on paper, but are lousy in practice)!

 

This sort of fossil hunting has become vendetta for me and I suspect I am using the wrong search terms in the academic data bases.

 

I'll be on an excursion until the 26th of July, but I'm going to try and check in here from Spanish Fork or Delta.

(And hopefully have some non-nebraska samples to work with and turn my students loose on!)

 

[Mediospirifer]

Just an amateur here, but I have some experience in the topic.

 

What kind of matrix are you working with? Acids work well for limestone or other carbonate minerals, but won't do much to non-carbonate rocks. If you put a chunk of matrix in acid and nothing happens, you need a different technique (Carbonates in acid will give off bubbles of CO₂ as they dissolve).

 

I haven't done much with matrices other than limestone and soft shale or clay. For the limestone, I use vinegar in an overnight soaking (repeated many times; it's slow!), and for the shale I use the freeze-thaw method. Clay and mud I wash with lots of water.

 

All of these techniques are slow. You may be able to speed the breakdown of shale or clay with some hydrogen peroxide (dry the matrix thoroughly, then add the H₂O₂). Or there are other chemicals that the real pros use. I've seen something called Rewoquat or Varisoft recommended for shales.

 

Want some good conodont matrix? I'd be happy to trade (or sell) a box of North Evans limestone chunks. 

 

[LabRatKing]

Thanks for the info! Lately I have been working with the various limestone and shale from Eastern Nebraska. When I return from my Utah trip, I'll have quite a few samples.

our limestone here is great at neutralizing vinegar concentrations of acetic acid. We've been working with concentrations from 10  - 35% to achieve a good digest. We place the tubs in a shaker/incubator at 30-40C to speed up the process, but it still can take 21 days!

 

I've been hesitant to try the formic acid method as I have developed a sensitivity to it thanks to previous, non fossil related projects.

 

I had not heard of using hydrogen peroxide as a catalyst, but I will definitely give it a try! It makes complete sense to me.

[Mediospirifer]

I wouldn't advise mixing acid with peroxide--or at least test a tiny amount separately first! 

 

It sounds like you have a higher-tech setup than I do. Do you have access to facilities for handling liquid nitrogen? I have a paper on using that to extract foraminifera from hard shale I could send you by PM. It might be too rough for conodonts, though.

 

[Acryzona]

I’ve used glacial acetic acid to recover conodonts from Indiana limestone, the formation escapes me at the moment.  I have some documentation on microfossil recovery with acetic acid solution, if you are interested please PM me.

 

For shale, I’ve had some success with soaking kerosene then soaking in water.  Repeating this cycle many times does breakdown the shale to release micros.

[LabRatKing]

13 hours ago, Mediospirifer said:

I wouldn't advise mixing acid with peroxide--or at least test a tiny amount separately first! 

 

It sounds like you have a higher-tech setup than I do. Do you have access to facilities for handling liquid nitrogen? I have a paper on using that to extract foraminifera from hard shale I could send you by PM. It might be too rough for conodonts, though.

 

I have access to full laboratory facilities ( I manage five labs at a university). I'd love to read up on the forams! I have been using (well attempting) to use microfossils to train students on operations of the scanning electron microscope.

[Mediospirifer]

5 hours ago, LabRatKing said:

I have access to full laboratory facilities ( I manage five labs at a university). I'd love to read up on the forams! I have been using (well attempting) to use microfossils to train students on operations of the scanning electron microscope.

 

Ah! Then you may want to get matrix that has pollen grains or diatoms, too. If you want to go really tiny. 

 

My experience with breaking down rocks is that it is a slow process. Shale that's weathered into mud is faster to process, since it's already broken down, but it can include more modern material than the source rock. 

 

I think the person here that would be most helpful to you (if he's around) is @MarcoSr. Hopefully, he'll check in soon!

 

PM sent. Enjoy the paper!

 

[MarcoSr]

15 hours ago, Mediospirifer said:

 

I think the person here that would be most helpful to you (if he's around) is @MarcoSr. Hopefully, he'll check in soon!

 

 

Since you have a laboratory environment, you might look into using lab grade 30% H2O2 to break down your shale.  I can only use 3% Walmart H2O2 in my home environment which does work somewhat on soft weathered shale.

 

I've always wanted to try liquid nitrogen but you can't do that in a home environment.

 

With some limestone and other carbonate materials combining freeze/thaw with vinegar (5% or 10% acetic acid) or mild acid like acetic acid in low concentrations (10%) speeds up the process.  Boil the limestone/carbonate material in vinegar/low concentration acetic acid, sieve it after cooling, put the bigger pieces of matrix in the freezer and wash the matrix residue that has broken down really well in water to remove any residual acid before searching/storage. Repeat the process until you get the amount of breakdown that you want. 

 

I never use strong acids or high concentrations of weak acids.  It is not safe to use them in a home environment and they destroy your fossil specimens.  These acids etch or may even dissolve the phosphate based vertebrate specimens (shark, ray, bony fish) that I'm trying to extract from the matrix.  Even weak acids in low concentrations will dissolve carbonate based invertebrates.

 

When using any of these methods, safety has to be your number one concern.  Don't even consider using 30% H2O2 or strong acids/weak acids in high concentration in a home environment.

 

Edit:  I should have said something about clay and/or sand matrix.  I take this type of matrix from different formations in VA/MD in 5 gallon buckets.  Once home, I put the matrix on tarps and dry it really well in the sun.  When thoroughly dry, I fill 5 gallon buckets about 1/3 full of matrix, add hot tap water and a few drops of detergent like Dawn dish soap, stir the matrix really well and then let the buckets sit overnight.  The next morning I wash the matrix with water in a sieve (size depends upon what I'm looking for but I usually don't go below .4 mm and usually use 1 mm.) which fits over a 5 gallon bucket.  I usually get about 90% breakdown on the first washing.  If I get good breakdown I put the remaining matrix on cookie pans or if there are still lots of small chunks I put the matrix back on the tarps and dry it again in the sun.  I repeat the above process as many times as necessary to get full breakdown.  Most clay and/or sand matrix breaks down completely in two or three cycles.

 

 

Marco Sr.

[LabRatKing]

Excellent! Thanks for all the info! I'm not a fan of cryogenic methods as they can be hard to control. I just got back with about 15 kilos of known Compton samples, so I'll give some of these tips a try.

one of the nice things about having full lab acess, is that I can do stuff no one should ever try at home.

I'll report back shortly!

[Johannes]

@LabRatKing : There is one definite book about working techniques in micropalaeontology:

 

Wissing & Herrig

 

Unfortunately in german and not translated into english.

 

For non-calcareous microfossils in carbonaceous rocks you can use HCl or CH3COOH, but conodonts get slightly corroded up to completly dissolved (esp. with HCl), if you didn't act carefull enough. Usually you have to buffer a 7% CH3COOH with e.g. C4H6CaO4. If you have marly limestones you have to go up to 20% CH3COOH, but you have take out the sample-residue very often (5-8h). You can also use CH2ClCOOH, if the limestones are slightly dolomitized. Alternatives are EDTA or HCOOH (10%) buffered with CaCO3 and Ca3(PO4)2. For bituminous shales  you can use H3PO4 and, for slightly silified hoststones HF is mandatory (CaPO4 of the conodonts turns into CaF2), but HF is slightly dangerous, even in a professional lab, as you know.

For marls you can use H2O2 (but I think than you destroy the "conodont-metamorphic-thermometer") or tensids like Rewoquad (wich is the less agressive chemical for microfossils you can use (see Lierl 1992 or Jarochowska et al. 2013)

 

Esp. for conodonts from larger samples (usually they can be quite rare) you need some floating techniques, if you need some further informations, please PM me.

 

Best regards

 

Johannes

[Johannes]

On 27.7.2019 at 9:02 PM, LabRatKing said:

I just got back with about 15 kilos of known Compton samples

 

Following my experience from devonian reef limestones, 15 kg can be a rather small amount of samples to get satisfying numbers of conodonts.

[Johannes]

1 minute ago, Johannes said:

 

double post, can be deleted

 

[LabRatKing]

2 hours ago, Johannes said:

@LabRatKing : There is one definite book about working techniques in micropalaeontology:

 

Wissing & Herrig

 

Unfortunately in german and not translated into english.

 

For non-calcareous microfossils in carbonaceous rocks you can use HCl or CH3COOH, but conodonts get slightly corroded up to completly dissolved (esp. with HCl), if you didn't act carefull enough. Usually you have to buffer a 7% CH3COOH with e.g. C4H6CaO4. If you have marly limestones you have to go up to 20% CH3COOH, but you have take out the sample-residue very often (5-8h). You can also use CH2ClCOOH, if the limestones are slightly dolomitized. Alternatives are EDTA or HCOOH (10%) buffered with CaCO3 and Ca3(PO4)2. For bituminous shales  you can use H3PO4 and, for slightly silified hoststones HF is mandatory (CaPO4 of the conodonts turns into CaF2), but HF is slightly dangerous, even in a professional lab, as you know.

For marls you can use H2O2 (but I think than you destroy the "conodont-metamorphic-thermometer") or tensids like Rewoquad (wich is the less agressive chemical for microfossils you can use (see Lierl 1992 or Jarochowska et al. 2013)

 

Esp. for conodonts from larger samples (usually they can be quite rare) you need some floating techniques, if you need some further informations, please PM me.

 

Best regards

 

Johannes

Excellent!

The good news is I can passably read German, so I ordered a copy.

 

Thanks for all the information! I brought a few samples to the labs with me today. Buffered EDTA is one I should have thought of, but didn't. Looking forward to trying it out.

I suspect a  big part of our issue is the dissolution of the specimens due to concentration and/or time in the bath.

[Johannes]

52 minutes ago, LabRatKing said:

I suspect a  big part of our issue is the dissolution of the specimens due to concentration and/or time in the bath.

 You need to preprep the samples. Cracking down the rocks into pieces of around 2-4cm in diameter, that will fasten the dissolution process*. It is usefull to hang the crushed rock samples with a wide-porous plastic sieve* into the reacting liquid, so chemoeroded material can sink to the ground without being harmed by overlying pressure of the sample material. Also doing it in this way it is easier to take it out of the agressive reactans and decant that material during the reaction process.

 

*you have to optimize the process for your specific sample material...

[LabRatKing]

3 hours ago, Johannes said:

 You need to preprep the samples. Cracking down the rocks into pieces of around 2-4cm in diameter, that will fasten the dissolution process*. It is usefull to hang the crushed rock samples with a wide-porous plastic sieve* into the reacting liquid, so chemoeroded material can sink to the ground without being harmed by overlying pressure of the sample material. Also doing it in this way it is easier to take it out of the agressive reactans and decant that material during the reaction process.

 

*you have to optimize the process for your specific sample material...

I have been experimenting a bit and have been using Imhoff sediment cones for collection, cell culture sieves and numerous grades of geology mesh sieves. I took your advice and pre-prepped some samples today and set up a few different digestion baths. Hopefully tomorrow I will have some images to share!

 

I really appreciate all the advice on this topic! Using prepped microfossils is much less hazardous than biological specimens for SEM training. (It is also far less expensive...tungsten/platinum/gold sputtering and uranyl acetate are expensive, whereas microfossils, once extracted, are ready to go under the gun relatively quickly!)

 

I have a few hundred liters of N2 on the way for the fall term, so I should be able to report back on the cryo methods in a few weeks.

[Mediospirifer]

I had a thought for something else that ought to give you some good samples for electron microscopy. Diatomaceous earth is a soft matrix with a high proportion of silicified diatom fossils. You probably wouldn't need to do more than (gently!) wash and sieve to find lots of cool specimens. It's available at any garden store, as a nontoxic pest killer.

 

For most of us on this forum, the biggest problem with a bag of garden-variety diatomaceous earth would be that it's probably going to be difficult to identify the source (and therefore the age) of the material. In your case, you and your students might not care about precisely identifying the fossils.

 

21 minutes ago, LabRatKing said:

I have a few hundred liters of N2 on the way for the fall term, so I should be able to report back on the cryo methods in a few weeks.

 

Great! I got that paper from MarcoSr, and haven't had anything I wanted to try LN2 on (I also work in a lab). I'd be interested in hearing about how well it works, and seeing pictures of what you find!

 

[LabRatKing]

18 minutes ago, Mediospirifer said:

I had a thought for something else that ought to give you some good samples for electron microscopy. Diatomaceous earth is a soft matrix with a high proportion of silicified diatom fossils. You probably wouldn't need to do more than (gently!) wash and sieve to find lots of cool specimens. It's available at any garden store, as a nontoxic pest killer.

 

For most of us on this forum, the biggest problem with a bag of garden-variety diatomaceous earth would be that it's probably going to be difficult to identify the source (and therefore the age) of the material. In your case, you and your students might not care about precisely identifying the fossils.

 

 

Great! I got that paper from MarcoSr, and haven't had anything I wanted to try LN2 on (I also work in a lab). I'd be interested in hearing about how well it works, and seeing pictures of what you find!

 

We actually started with diatomaceous earth, and it is excellent for training the students on operation of the SEM, but eventually, I have to get these kids using wonderfully dangerous stuff like uranium and osmium, so I figure that some basic chemistry and geology prep is a good next step. We are a bit odd as universities go as we insist our undergrads learn how to use all the equipment prior to graduation...Plus we are so small that I get to wear the TA hat along with the Lab manager hat, lead tech hat, and PI hat on my own research. Hopefully they won't make me teach actual lecture courses too!

[Mediospirifer]

35 minutes ago, LabRatKing said:

We actually started with diatomaceous earth, and it is excellent for training the students on operation of the SEM, but eventually, I have to get these kids using wonderfully dangerous stuff like uranium and osmium, so I figure that some basic chemistry and geology prep is a good next step. We are a bit odd as universities go as we insist our undergrads learn how to use all the equipment prior to graduation...Plus we are so small that I get to wear the TA hat along with the Lab manager hat, lead tech hat, and PI hat on my own research. Hopefully they won't make me teach actual lecture courses too!

 

Ah, that makes sense. So the lessons are not just about the SEM, it's about various preparation techniques. Cool!

 

I'll repeat my offer for some North Evans limestone, if you're still looking for conodonts. That's not a reef deposit, it's a beach lag deposit. Johannes said above, most limestones are pretty sparse in conodonts. NELS is rich. On my first attempt at dissolving pieces, I placed a bunch of chunks (1" cube average) in vinegar overnight (4 pint-sized containers), and rinsed them and filtered the residue the following day. I found 6 conodonts in the largest sieve fraction from that residue. Smaller fractions also have them, but I had several days residue mixed together before I started seriously searching those. I've found several hundred conodonts in total, and I don't think I've dissolved more than a fraction of a kilogram of rock.

 

The trick is to not be in a hurry. I fit as much rock as possible into a container, then add vinegar, and wash the residue daily. It takes a few days before the rock is visibly (without magnification) etched (although I can feel the change in surface texture as the microfossils are exposed), but I haven't seen any dissolution of my phosphatic fossils.

 

Let me know if you're interested. I should revisit the matrix anyway.